The Protoperithecia Assay quantifies photoperiodic responses in Neurospora crassa by measuring protoperithecia formation under defined light–dark cycles. This plate-based method provides a simple and scalable approach for comparing photoperiod-dependent developmental responses across experimental conditions.
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Method Article
Plate-Based Imaging Assay for Quantifying Photoperiodic Responses in Neurospora crassa Using Protoperithecia Development
In This Article
Summary
Abstract
Neurospora crassa is a well-established model organism for studying biological timing and light-regulated development. The Protoperithecia Assay (PPA) is a plate-based imaging method for quantifying photoperiodic responses by measuring protoperithecia formation as a developmental output. The goal of this protocol is to provide a reproducible and scalable workflow for assessing how defined light–dark cycles influence fungal sexual development. In this assay, fungal cultures are grown on synthetic crossing medium under controlled photoperiod conditions, followed by standardized removal of surface conidia to improve visualization. Protoperithecia are quantified by manual image-based counting, and average counts per plate are used for statistical comparisons across photoperiod conditions. Unlike prior approaches, the PPA integrates standardized culture conditions, controlled photoperiod exposure, and quadrant-based imaging to enable reproducible quantitative comparisons across strains. This workflow extends beyond a basic protocol by establishing a consistent and scalable framework for measuring photoperiod-dependent developmental output under defined experimental conditions. The method requires approximately one week from inoculation to analysis and can be applied across multiple strains under defined environmental conditions. The PPA provides a simple and accessible platform for comparative analysis of photoperiod-dependent development in fungi under controlled laboratory conditions.
Introduction
The protoperithecia assay (PPA) quantifies photoperiodic responses in the fungus Neurospora crassa. Many organisms measure day and night length to coordinate biological processes in a phenomenon known as photoperiodism1,2,3,4,5,6,7,8. In this assay, the development of the female sexual structure, the protoperithecium, serves as a measurable, light-dependent proxy for photoperiodic response.....
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Protocol
The reagents, software, and equipments used are listed in the Table of Materials.
1. Preparation of media and materials
(Estimated time: 1–1.5 h active time; overnight cooling optional)
- Prepare synthetic crossing (SC) media
- Prepare SC medium: 0.2% sucrose, 5 µg/L biotin, 1.5% agar, and Westergaard’s salts (pH 6.5 with 1 N NaOH)15.
- Autoclave the medium. Allow it to cool to 50–55 °C until it reaches a pourable state.
CAUTION: Use heat-resistant gloves when removing....
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Results
Following inoculation with the wild-type strain, conidia covered the SC surface within 36–48 h. After 7 days of photoperiod exposure, a dense conidial layer formed and was removed with a moistened paper towel saturated with 70% ethanol (Figure 1). This step exposed underlying protoperithecia without disrupting their structure. Under microscopy, protoperithecia appeared as dark, circular structures distributed across the plate surface. These structures exhibited a spherical morphology and wer.......
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Discussion
The goal of the PPA is to use sexual development in fungi as a proxy for determining whether an organism perceives a specific photoperiod. By quantifying the average number of protoperithecia produced under different light cycles, photoperiodic responses can be assessed and compared across conditions. Consistent with previous observations4,16, N. crassa exhibits higher protoperithecia production under equinox-like photoperiods, whereas short-day conditio.......
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Disclosures
The authors declare no competing interests.
Acknowledgements
The authors thank Cathyrn Maienza, Myo Thinzar Htin Aung, Aye Thinzar Htin Aung, and Morgan Bartleson for their support, assistance, and contributions to this project. The authors also acknowledge the Department of Biology at Rutgers University–Camden and the Center for Computational and Integrative Biology (CCIB) for providing institutional support, research facilities, and resources.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Agar | BD | 90000-786 | For Synthetic Crossing Media/Minimal Media |
| Alcohol burner flame | Wards | 470346-104 | For sterilizing inoculation loop |
| Ammonium iron sulfate hexahydrate | Supelco | FX0245-1 | For Synthetic Crossing Media/Minimal Media |
| Ammonium nitrate (NH4NO3) | BeanTown Chemical | BT134170-250G | For Minimal Media |
| Autoclave | VWR International | 76683-456 | For sterilizing glassware and media |
| Biological Safety Cabinet | ThermoFischer Scientific | 1323TS | For reduccing contamination of the plates, and the lab. |
| Biotin | VWR | 97061-444 | For Synthetic Crossing Media |
| Boric acid (H3BO3) | Electron Microscopy Sciences | 100503-696 | For Synthetic Crossing Media/Minimal Media |
| Calcium chloride (CaCl2) | AVANTOR PERFORMANCE MATERIAL LLC | JT1311-1 | For Synthetic Crossing Media |
| Calcium chloride dihydrate (CaCl2·2H2O) | Sigma-Aldrich | 223506-25G | For Minimal Media |
| Citric acid monohydrate (C6H8O7·H2O) | Sigma-Aldrich | 1002440500 | For Synthetic Crossing Media/Minimal Media |
| Copper sulfate pentahydrate (CuSO4·5H2O H2O) | BeanTown Chemical | BT121245-1KG | For Synthetic Crossing Media/Minimal Media |
| Ethanol | Decon Labs | 71001-754 | Used to remove conidia from plate surface and sterilizing inoculation loop |
| FIJI/ImageJ | National Institutes of Health (NIH) | N/A | For analyzing images/counting |
| Gloves | SW Safety Solutions | 76382-944 | For safety |
| GraphPad Prism | GraphPad Software, LLC | N/A | For performing statistical tests and creating figures |
| Growth Chamber | Caron Products & Services | 6320 & 6340 Series | Programmable for photoperiod cycles |
| HOBO Data Logger | Onset Computer Corporation | MX2202 | For light intensity and temperature logging |
| Inoculation loop | VWR International | 10806-354 | For transferring fungal material to petri dishes |
| Magnesium sulfate heptahydrate (MgSO4·7H2O) | BeanTown Chemical | BT145380-1KG | For Synthetic Crossing Media/Minimal Media |
| Manganese sulfate monohydrate (MnSO4·H2O H2O) | Sigma-Aldrich | 1059410250 | For Synthetic Crossing Media/Minimal Media |
| Microscope (Leica S6 D Greenough Stereo Microscope) | Leica Microsystems | 10446297 | For imaging |
| Neurospora crassa FGSC 2489 | Fungal Genetics Stock Center | 74-OR23-1VA, JCM19069 | Fungal strain |
| Paper Towels | Kimberly-Clark Professional, Scott and Kleenex | 6666114 | For pouring media |
| Permanent Marker | Sharpie | 30072 | For labeling plates |
| Petri Dishes | VWR International | 25384-164 | For plating Synthetic Crossing Media |
| Pipettes | VWR International | 75816-090 | For pouring media |
| Potassium dihydrogen phosphate (KH2PO4) | BeanTown Chemical | BT225070-500G | For Synthetic Crossing Media/Minimal Media |
| Potassium nitrate (KNO3) | Sigma-Aldrich | 221295-100G | For Synthetic Crossing Media |
| Small Disposable Glass Test Tubes | VWR International | 470211-698 | For initial fungal strain growth |
| Sodium chloride (NaCl) | VWR International | BDH9286-500G | For Synthetic Crossing Media |
| Sodium citrate dihydrate (Na3C6H5O7·2H2O) | VWR | 97061-008 | For Minimal Media |
| Sodium hydroxide (NaOH) | AVANTOR PERFORMANCE MATERIAL LLC | JT5635-2 | For Synthetic Crossing Media |
| Sodium molybdate dihydrate (Na2MoO4·2H2O) | Supelco | 1.06524.1000 | For Synthetic Crossing Media/Minimal Media |
| Sucrose | VWR International | BDH9308-500G | For Synthetic Crossing Media/Minimal Media |
| Zinc sulfate heptahydrate (ZnSO4·7H2O) | BeanTown Chemical | BT122545-500G | For Synthetic Crossing Media/Minimal Media |
References
- Wood SH et al. Circadian clock mechanism driving mammalian photoperiodism. Nat Commun. 2020;11:4291.
- Wikelski M et al. Avian circannual clocks: adaptive significance and possible involvement of energy turnover in their proximate control. Philos Trans R Soc Lond B Biol Sci. 2008;363:411-23.
- Wang Q et al. Plants distinguish different photoperiods to independently control seasonal flowering and growth. Science. 2024;383:eadg9196.
- Tan Y, Merrow M, Roenneberg T. Photoperiodism in Neurospora crassa. J Biol Rhythms. 2004;19:135-43.
- Kumar V. Photoperiodism in higher vertebrates: an adaptive strategy in temporal environment. Indian J Exp Biol....
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